Rapid processing of information in the nervous system requires generation and saltatory conduction of action potentials along myelinated axons. In excitable cells, voltage-gated Na+ channels are responsible for action potential conduction and propagation. In myelinated axons, Na+ channels localize at high concentration in gaps between the myelin sheaths called nodes of Ranvier. Na+ channels are composed of a pore-forming a subunit and one or two betaa subunits. It is hypothesized that beta subunits serve as critical links between the extracellular environment and intracellular signal transduction, especially in the axo-glial apparatus where they may function to mark presumptive nodes and participate in axo-glial communication. Na+ channel beta1 and beta2 subunits are multifunctional. In addition to modulation of ion channel function and cell surface expression, beta1 and beta2 act as cell adhesion molecules of the immunoglobulin superfamily and participate in homophilic and heterophilic cell adhesion, interaction with extracellular matrix molecules, and interaction with the cytoskeleton. In the present exploratory grant application, it is proposed to develop the zebrafish (zf) system to identify zf beta subunits and to study Na+ channel a and a subunit targeting during nodal formation. The long-term goal will be to use reverse genetics to knockdown zf beta1 and beta2 expression to determine their roles in nodal formation. The proposed activities will lay the foundation for future success with this model system. Isoforms of zf beta1 and beta2 have been identified that are different from those previously cloned from mice. Thus, these studies may lead to significant advances in Na+ channel biology that were not thought to be possible using mouse models. The following specific aims are proposed: 1. To test whether novel a subunits identified in zf display unique subcellular localization, alpha subunit association, electrophysiological properties, or cell adhesive properties. 2. To investigate the temporal order of Na+ channel alpha and beta subunit targeting to nodes of Ranvier. Using transgenes encoding fluorescent-tagged zfNav1.6 and zf beta subunits under control of the zf-tubulin promoter, the temporal order of Na+ channel subunit localization to zf optic nerve nodes of Ranvier during development will be examined. Transgenes expressing labeled forms of neurofascin186, contactin, and ankyrinG will then be used to determine the temporal order of Na+ channel subunit appearance relative to these nodal proteins. Development of this powerful genetic approach will make a number of significant contributions to the understanding of the formation and maintenance of CNS nodes of Ranvier, as similar experiments in mouse models have not yet been possible. This information will be critical to the future development of therapies to treat diseases involving neuronal hyperexcitability such as epilepsy or diseases involving action potential conduction blockade such as Multiple Sclerosis.